Subtype Of Non-small Cell Lung Cancer Research Articles

Abstract 4906: Association between low-frequency pretreatment EGFR T790M mutation and tumor immune microenvironment in patients with EGFR-mutated non-small cell lung cancer

Abstract Background: Involvement of tumor immune microenvironment including programmed death 1 (PD-1) and PD-ligand 1 (PD-L1) in biological properties of EGFR-mutant non-small cell lung cancer (NSCLC) remains elusive. Correlation of EGFR mutational status and PD-L1 expression and its impact on anti-PD-1/PD-L1 therapies have been intensively investigated but the relationship between pretreatment EGFR T790M (preT790M) and PD-L1 status has not yet been tackled. We have previously reported that EGFR-TKI-naïve patients with high preT790M had significantly shorter progression-free survival (PFS) upon EGFR-TKI treatment. In this study we investigated the association between preT790M status of tumor and immune microenvironment and its impact on efficacy of EGFR-TKI. Materials and methods: We had previously assessed preT790M by droplet digital PCR in 44 advanced NSCLC patients harboring activating EGFR mutations treated with first-line EGFR-TKIs at Osaka City University Hospital between August 2013 and July 2016. For the current study, tumor proportion score (TPS) for PD-L1 and PD-L2 expression was assessed immunohistochemically using 28-8 antibody and D7U8C antibody, respectively. CD8-positive tumor-infiltrating lymphocytes (TILs) in tumor specimens were also evaluated. Results: PD-L1/PD-L2 TPS and CD8-positive TILs were evaluable in 39 of 44 pretreatment tumor specimens. PD-L1 TPS of ≥50%, 1-49% and <1% were observed in 9 (23.1%), 10 (25.6%) and 20 patients (51.3%), respectively. PD-L2 TPS of ≥50%, 1-49% and <1% were observed in 1 (2.6%), 8 (20.5%) and 30 patients (76.9%), respectively. Significantly more PD-L1 high cases (TPS of ≥50%) were detected in subgroup of tumor with preT790M (41.2%, n=7/17) than in that without preT790M (9.1%, n=2/22) (p=0.026). In non-preT790M subgroup, CD8-positive TILs density was significantly higher in PD-L1 high than in PD-L1 low (median: 1298/mm2 vs. 331/mm2, p=0.035), while in preT790M subgroup, there was no significant difference regardless of PD-L1 level (median: 329/mm2 vs. 226/mm2, p=0.19). The median PFS and response rate for initial EGFR-TKIs were 13.6 months and 95% in non-preT790M/PD-L1 low, 1.6 months and 0% in non-preT790M/PD-L1 high, 11.4 months and 80% in preT790M/PD-L1 low, and 6.7 months and 57% in preT790M/PD-L1 high, respectively. No correlation was observed between preT790M and PD-L2 expression levels. Conclusion: Results of our study suggest that there may exist the subtypes in EGFR-mutated NSCLC according to PD-L1 status and tumor immune microenvironment. Differential efficacy of EGFR-TKIs based on these subtypes further implies the potential impact of PD-L1 status and tumor immune microenvironment in EGFRblockade along with preT790M. Citation Format: Yoshiya Matsumoto, Kenji Sawa, Mitsuru Fukui, Jun Oyanagi, Motohiro Izumi, Koichi Ogawa, Tomohiro Suzumura, Tetsuya Watanabe, Hiroyasu Kaneda, Shigeki Mitsuoka, Kazuhisa Asai, Tatsuo Kimura, Nobuyuki Yamamoto, Tomoya Kawaguchi, Yasuhiro Koh. Association between low-frequency pretreatment EGFR T790M mutation and tumor immune microenvironment in patients with EGFR-mutated non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4906.

Abstract 3214: In situ vaccination with CCL21-modified dendritic cells (CCL21-DC) combined with checkpoint blockade in murine models of NSCLC

Abstract Studies that target the PD-1/PD-L1 axis of immune checkpoints have demonstrated clinical responsesin approximately 20% of non-small cell lung cancer (NSCLC) patients.However, majority of the patients do not benefit from the initial treatment.Mechanistic studies reveal that responses to PD-1/PD-L1 blockade are associated with high tumor mutational burden (TMB) andincreased CD8+T cell tumor infiltration and high baseline tumor PD-L1 expression. Furthermore, recent studies have identified Stk11/Lkb1 loss as a major driver of primary resistance to PD-1 blockade in inKRAS-mutant lung adenocarcinoma (LUAC), a major subtype of NSCLC. One potential approach to enhance the effectiveness of checkpoint inhibitors is to enhance tumor antigen presentation and tumor-specific T cell immune responses by in situvaccination, taking advantage of the full repertoire of available tumor antigens. In preclinical and clinical studies, we have demonstrated that CCL21-secreting dendritic cells (CCL21-DC) have the capacity to induce the infiltration of DC and T cells into the TME and to promote tumor-specific T cell activation both locally and systemically. This response, however, was accompanied by the upregulation of PD-L1 expression in the tumor. We hypothesize that in situvaccination with CCL21-DC could restore tumor antigen presentation and T lymphocyte infiltration into the tumor, thereby sensitizing non-responsive NSCLC tumors to checkpoint blockade. To test this hypothesis, we first established novel genetically-engineered murine models (GEMMs) of lung cancer that bear the common driver mutations (KPL; KrasG12D;Tp53-/-;Lkb1-/-) and varying mutational loads to accurately reflect the clinical disease. This was achieved by exposing KPL cells in vitroto the tobacco carcinogen N-methyl-N-nitrosourea (MNU) with various duration. Using these models, we demonstrated that (1) KPL with higher TMB is more sensitive to anti-PD-1 monotherapy as compare to low TMB, consistent with previous studies; (2) Intratumoral (IT) administration of CCL21-DC in combination with anti-PD-1 resulted in significantly enhanced anti-tumor response compared to either monotherapy alone. Further work is underway to evaluate the efficacy of the combination therapy with IT CCL21-DC and PD-1 inhibition in multiple murine models of NSCLC. In addition, mechanistic studies will be performed to understand the anti-tumor effect mediated by the CCL21-DC and anti-PD-1 combination therapy, including immune phenotyping of TME and functional studies of DC and T cells. As we prepare to initiate our clinical trial that combines IT CCL21-DC with PD-1 inhibition in advanced NSCLC patients with low baseline PD-L1 expression, we anticipate these preclinical models will serve as a platform to enhance our understanding of the molecular mechanisms of response and resistance to immunotherapy. Citation Format: Raymond J. Lim, Ramin Salehirad, Bin Liu, Rui Li, Linh M. Tran, Kostyantyn Krysan, Stephanie Ong, Zi L. Huang, David B. Shackelford, Sherven Sharma, Steven M. Dubinett. In situ vaccination with CCL21-modified dendritic cells (CCL21-DC) combined with checkpoint blockade in murine models of NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3214.

Identification of differentially expressed genes between lung adenocarcinoma and squamous cell carcinoma using transcriber signature analysis

To analyze the differentially expressed genes (DEGs) between lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) with bioinformatics analysis and search for potential biomarkers for clinical diagnosis of nonsmall cell lung cancer (NSCLC). The gene expression profiling datasets of LUAD and LUSC were acquired. The transcriptome differences between LUAD and LUSC were identified using R language processing and t-test analysis. The differential expressions of the genes were shown by Venn diagram. The DEGs identified by GEO2R were analyzed with DAVID and Ingenuity Pathway Analysis (IPA) to identify the signaling pathways and biomarkers that could be used for differential diagnosis of LUAD and LUSC. The TCGA data and the biomarker expression data from clinical lung cancer samples were used to verify the differential expressions of the Osteoarthritis pathway and LXR/RXR between LUAD and LUSC. We further examined the differential expressions of miR-181 and its two target genes, WNT5A and MBD2, in 23 clinical specimens of lung squamous cell carcinoma and the paired adjacent tissues. GEO data analysis identified 851 DEGs (including 276 up-regulated and 575 down-regulated genes) in LUAD and 885 DEGs (including 406 up-regulated and 479 down-regulated genes) in LUSC. DAVID and IPA analysis revealed that leukocyte migration and inflammatory responses were more abundant in LUAD than in LUSC. Osteoarthritis pathway was inhibited in LUAD and activated in LUSC. IPA analysis showed that transcription factors (GATA4, RELA, YBX1, TP63 and MBD2), cytokines (WNT5A and IL1A) and microRNAs (miR-34a, miR-181b and miR-15a) differed significantly between LUAD and LUSC. miR-34a with IL-1A, miR-15a with YBX1, and miR-181b with WNT5A and MBD2 could serve as the paired microRNA and mRNA targets for differential diagnosis of NSCLC subtypes. Analysis of the clinical samples showed an increased expression of miR-181b-5p and the down-regulation of WNT5A, which could be used as molecular markers for the diagnosis of LUSC. Through transcriptome analysis, we identified candidate genes, paired microRNAs and pathways for differentiating LUAD and LUSC, and they can provide novel differential diagnosis and therapeutic strategies for LUAD and LUSC.

Resistance mechanisms and potent-targeted therapies of ROS1-positive lung cancer.

The discovery of targetable mutations, which cause gene rearrangement, led to a major advancement in the treatment of patients with non-small cell lung cancer (NSCLC), and cancers with such mutations can be paired with drugs which specifically target them. c-ros oncogene (ROS1) positive NSCLC is one molecular subtype of NSCLC with a therapeutic target. Currently, different targeted therapies and ROS1 inhibitors have been discovered, but all are in different investigational phases, with only one (crizotinib) which is FDA approved. Crizotinib is a small-molecule tyrosine kinase inhibitor (TKI) which was discovered to actively inhibit ALK, MET, and ROS1. Crizotinib has shown to be remarkably efficacious against ROS1 lung cancer, prompting ROS1 detection in lung cancer to be quite significant. Sadly, crizotinib resistance in ROS1 is a frequent occurrence which poses a major clinical challenge in the successful treatment of ROS1 lung cancer; hence, the discovery of the second and third generation ROS1 inhibitors is of utmost importance. In this review, we discuss the underlying mechanisms through which ROS1 tumor cells acquire resistance to crizotinib-the first-line drug for ROS1-positive NSCLC, and summarize various new potent drugs which can overcome this resistance and serve as viable alternatives.

Comparing progression molecular mechanisms between lung adenocarcinoma and lung squamous cell carcinoma based on genetic and epigenetic networks: big data mining and genome-wide systems identification

Non-small-cell lung cancer (NSCLC) is the predominant type of lung cancer in the world. Lung adenocarcinoma (LADC) and lung squamous cell carcinoma (LSCC) are subtypes of NSCLC. We usually regard them as different disease due to their unique molecular characteristics, distinct cells of origin and dissimilar clinical response. However, the differences of genetic and epigenetic progression mechanism between LADC and LSCC are complicated to analyze. Therefore, we applied systems biology approaches and big databases mining to construct genetic and epigenetic networks (GENs) with next-generation sequencing data of LADC and LSCC. In order to obtain the real GENs, system identification and system order detection are conducted on gene regulatory networks (GRNs) and protein-protein interaction networks (PPINs) for each stage of LADC and LSCC. The core GENs were extracted via principal network projection (PNP). Based on the ranking of projection values, we got the core pathways in respect of KEGG pathway. Compared with the core pathways, we found significant differences between microenvironments, dysregulations of miRNAs, epigenetic modifications on certain signaling transduction proteins and target genes in each stage of LADC and LSCC. Finally, we proposed six genetic and epigenetic multiple-molecule drugs to target essential biomarkers in each progression stage of LADC and LSCC, respectively.

DNA methylation profiling reliably distinguishes pulmonary enteric adenocarcinoma from metastatic colorectal cancer

Pulmonary enteric adenocarcinoma is a rare non-small cell lung cancer subtype. It is poorly characterized and cannot be distinguished from metastatic colorectal or upper gastrointestinal adenocarcinomas by means of routine pathological methods. As DNA methylation patterns are known to be highly tissue specific, we aimed to develop a methylation-based algorithm to differentiate these entities. To this end, genome-wide methylation profiles of 600 primary pulmonary, colorectal, and upper gastrointestinal adenocarcinomas obtained from The Cancer Genome Atlas and the Gene Expression Omnibus database were used as a reference cohort to train a machine learning algorithm. The resulting classifier correctly classified all samples from a validation cohort consisting of 680 primary pulmonary, colorectal and upper gastrointestinal adenocarcinomas, demonstrating the ability of the algorithm to reliably distinguish these three entities. We then analyzed methylation data of 15 pulmonary enteric adenocarcinomas as well as four pulmonary metastases and four primary colorectal adenocarcinomas with the algorithm. All 15 pulmonary enteric adenocarcinomas were reliably classified as primary pulmonary tumors and all four metastases as well as all four primary colorectal cancer samples were identified as colorectal adenocarcinomas. In a t-distributed stochastic neighbor embedding analysis, the pulmonary enteric adenocarcinoma samples did not form a separate methylation subclass but rather diffusely intermixed with other pulmonary cancers. Additional characterization of the pulmonary enteric adenocarcinoma series using fluorescence in situ hybridization, next-generation sequencing and copy number analysis revealed KRAS mutations in nine of 15 samples (60%) and a high number of structural chromosomal changes. Except for an unusually high rate of chromosome 20 gain (67%), the molecular data was mostly reminiscent of standard pulmonary adenocarcinomas. In conclusion, we provide sound evidence of the pulmonary origin of pulmonary enteric adenocarcinomas and in addition provide a publicly available machine learning-based algorithm to reliably distinguish these tumors from metastatic colorectal cancer.

Profile of alectinib for the treatment of ALK-positive non-small cell lung cancer (NSCLC): patient selection and perspectives.

Discovered in 2007, anaplastic lymphoma kinase (ALK) gene rearrangements positive (ALK+) lung cancers compose a small subset of non-small cell lung cancer (NSCLC), with rapidly expanded treatments. There are currently several ALK inhibitors, including crizotinib, ceritinib, alectinib, brigatinib, and lorlatinib which have been licensed by the US Food and Drug Administration or the European Medicines Agency for the treatment of ALK+ NSCLC patients. Along with the multiple therapies, the survival of this subtype of NSCLC has been significantly expanded, even for patients whose disease has spread in the brain. Alectinib (Alecensa), a specific ALK and rearranged during transfection tyrosine kinase inhibitor is approved as first-line therapy for metastatic ALK+ NSCLC patients. It is additionally approved for ALK+ NSCLC previously treated with crizotinib. The main aim of this review is to assemble on the efficacy of alectinib for the treatment of ALK+ NSCLC, to elaborate the activity of the drug in the central nervous system, and to debate on which is the position of this compound in the treatment course of ALK+ lung cancer patients.

Farnesoid X Receptor Constructs an Immunosuppressive Microenvironment and Sensitizes FXRhighPD-L1low NSCLC to Anti-PD-1 Immunotherapy.

The farnesoid X receptor (FXR) regulates inflammation and immune responses in a subset of immune-mediated diseases. We previously reported that FXR expression promotes tumor cell proliferation in non-small cell lung cancer (NSCLC). Here we study the relevance of FXR to the immune microenvironment of NSCLC. We found an inverse correlation between FXR and PD-L1 expression in a cohort of 408 NSCLC specimens; from this, we identified a subgroup of FXRhighPD-L1low patients. We showed that FXR downregulates PD-L1 via transrepression and other mechanisms in NSCLC. Cocultured with FXRhighPD-L1low NSCLC cell lines, effector function and proliferation of CD8+ T cell in vitro are repressed. We also detected downregulation of PD-L1 in FXR-overexpressing Lewis lung carcinoma (LLC) mouse syngeneic models, indicating an FXRhighPD-L1low subtype in which FXR suppresses tumor-infiltrating immune cells. Anti-PD-1 therapy was effective against FXRhighPD-L1low mouse LLC tumors. Altogether, our findings demonstrate an immunosuppressive role for FXR in the FXRhighPD-L1low NSCLC subtype and provide translational insights into therapeutic response in PD-L1low NSCLC patients treated with anti-PD-1. We recommend FXRhighPD-L1low as a biomarker to predict responsiveness to anti-PD-1 immunotherapy.

Circulating tumor DNA detection is correlated to histologic types in patients with early-stage non-small-cell lung cancer

ObjectivesCirculating tumor DNA (ctDNA) testing in plasma in patients with non-small-cell lung cancer (NSCLC) has the potential to be a supplemental or surrogate tool for tissue biopsy. Detection of genomic abnormalities in ctDNA and their association with clinical characteristics in early-stage NSCLC need to be clarified. Materials and methodsHere, we comprehensively analyzed gene variations of 48 tumor tissues and 48 matched preoperative (pre-op) plasma and 25 postoperative (post-op) plasma from early-stage NSCLC patients using a targeted 546 genes capture-based next generation sequencing (NGS) assay. ResultsIn early-stage NSCLC, the average mutation allele frequency (MAF) in pre-op plasma ctDNA was lower than that in tissue DNA (tDNA). The concordant gene variations between pre-op ctDNA and tDNA were difficult to detect. However, we found the tissue- pre-op plasma concordant ctDNA mutation detection ratio in lung squamous cell carcinoma (LUSC) was much higher than that in lung adenocarcinoma (LUAD). We also established a LUSC-LUAD classification model by a least absolute shrinkage and selection operator (LASSO) based approach to help separate LUAD from LUSC based on ctDNA profiling. This model included 14 gene mutations and extracted an accuracy of 89.2% in the training set and 91.5% in the testing set. Correlation analysis showed tDNA-ctDNA concordant ratio was related to histologic subtype, gene mutations and tumor size in early-stage NSCLC. ConclusionThis study suggests histology subtype and gene mutations could affect ctDNA detection in early-stage NSCLC. NGS-based ctDNA profile has the potential utility in LUSC-LUAD classification.

Metabolic reprogramming and Notch activity distinguish between non-small cell lung cancer subtypes

BackgroundPrevious studies suggested that the metabolism is differently reprogrammed in the major subtypes of non-small cell lung cancer (NSCLC), squamous cell carcinomas (SCC) and adenocarcinomas (AdC). However, a comprehensive analysis of this differential metabolic reprogramming is lacking.MethodsPublicly available gene expression data from human lung cancer samples and cell lines were analysed. Stable isotope resolved metabolomics were performed on SCC and ADC tumours in human patients and in freshly resected tumour slices.ResultsAnalysis of multiple transcriptomics data from human samples identified a SCC-distinguishing enzyme gene signature. SCC tumours from patients infused with [U-13C]-glucose and SCC tissue slices incubated with stable isotope tracers demonstrated differential glucose and glutamine catabolism compared to AdCs or non-cancerous lung, confirming increased activity through pathways defined by the SCC metabolic gene signature. Furthermore, the upregulation of Notch target genes was a distinguishing feature of SCCs, which correlated with the metabolic signature. Notch and MYC-driven murine lung tumours recapitulated the SCC-distinguishing metabolic reprogramming. However, the differences between SCCs and AdCs disappear in established cell lines in 2D culture.ConclusionsOur data emphasise the importance of studying lung cancer metabolism in vivo. They also highlight potential targets for therapeutic intervention in SCC patients including differentially expressed enzymes that catalyse reactions in glycolysis, glutamine catabolism, serine, nucleotide and glutathione biosynthesis.

The prognostic value of KRAS mutation subtypes and PD-L1 expression in patients with lung adenocarcinoma.

e20022 Background: KRAS gene mutations are found in 20-30% of non-small cell lung cancer (NSCLC), especially in adenocarcinoma. Compared to EGFR and ALK mutations, KRAS mutation in NSCLC may be associated with poorer prognosis. However, prognostic value of different KRAS mutation subtypes in NSCLC remains unknown. The goal of this study is to evaluate the association of KRAS mutation subtypes, PD-L1 expression and clinical outcome in patients diagnosed with primary lung adenocarcinoma. Methods: A total of 256 patients diagnosed with KRAS mutated (determined by pyrosequencing) lung adenocarcinoma between 2011 and 2015 in AdventHealth-Orlando were evaluated for their cancer staging, Relapse-free survival (RFS) and overall survival (OS). PD-L1 staining was performed in a subgroup of 95 patients from the same cohort. PD-L1 expression was determined with immunohistochemistry staining using Ventana PD-L1 (SP263) assay. Results: In this cohort of patients, 65 had stage I disease, 29 stage II, 49 stage III, 110 stage IV and 3 with no clinical stage information; 104 male, 152 female; median age 67. All patients received standard surgical/chemo/radio therapy according to the disease stages. Only one single patient received immunotherapy. Nine subtypes of KRAS mutation were detected, predominantly in codon 12. Three major subtypes account for 83.6% KRAS mutations, namely G12C (45.7%), G12V (21.9%) and G12D (16.0%). We found that these three major subtypes had no difference in cancer stages (P = 0.58) or brain metastasis at diagnosis (P = 0.78). Kaplan Meier analysis also showed similar OS and RFS among all KRAS subtype. 34% of KRAS mutated patients were found to be PD-L1 positive with tumor proportion score of greater than or equal to 1. PD-L1 expression status was indistinct among the three major mutation subtypes (P = 0.38). Of interest, among patients with G12C mutation, positive PD-L1 expression was associated with significantly shorter OS (5.7 vs 12.8 months; P = 0.007). In contrast, for patients with other KRAS mutation subtypes, PD-L1 expression status had no impact on OS (P = 0.63). Conclusions: The clinical courses, including tumor stage at diagnosis, presence of brain metastasis, OS and RFS, are similar among lung adenocarcinoma patients with different KRAS mutation subtypes. Additionally, PD-L1 expression status appears to be independent of KRAS mutation subtypes. Of note, concurrent PD-L1 expression and G12C mutation is associated with particularly poorer prognosis. Further study is needed to see if PD1/PD-L1 block may improve outcome of this group of patients.

NSCLC mutation frequency in the central American and Caribbean region.

e14278 Background: Lung Cancer (LC) constitutes a leading cause of death in the World. Non-small cell lung cancer subtype adenocarcinoma (NSCLC-A) has been associated with epidermal growth factor receptor (EGFR) mutations, which is essential for treatment decisions. The EGFR mutation frequency has been proven to have regional variability in this NSCLC-A population. The current study presents the EGFR mutation incidence and T790M frequency in NSCLC-A cases diagnosed in Central American and Caribbean(CA&C) region. Methods: A cross-sectional evaluation was performed in patients diagnosed with NSCLC-A between 2016 and 2018 in five CA&C countries. Patients were obtained from Reference Centers in Dominican Republic, Nicaragua, Panama, Honduras and Costa Rica through the AstraZeneca testing program. EGFR mutations were evaluated through Real-Time PCR. Results: The research included 972 cases. Mean of age at the diagnosis was 62.79(28.12-90.46) years. Overall distribution of cases by gender was female in 52.37% (509/972) and male in 47.64% (463/972). For the group of antiEGFR TKI naïve patients, tissue samples were processed in 67.83% (601/886). For the group of post antiEGFR TKI patients plasma samples were processed in 90.70% (78/86). For the group of antiEGFR TKI naïve patients, EGFR mutations were present in 26.64% (236 / 886), the mean of age was 63.17 years (28.12-113.47). The frequency of Exon 19 was 53.38(126/236)% and Exon 21 was 27.54%(65/236)% and 11.01% (26/236) were complex mutations. T790M mutations in this treatment naïve population was 2.14%(19/886). For the group of post antiEGFR TKI patients the frequency of T790M mutation was 50.00% (43/86) where 78.57% were obtain from plasma samples. Conclusions: The frequency of NSCLC EGFR mutation positive patients in the CA&C region was similar to that previously described in other Latin American (LA) countries. In the treatment naïve population, tissue samples remain the most important sample to analyze for EGFR mutations. T790M mutations can be seen in treatment naïve population. For patients that progressed to an antiEGFR TKI, T790M mutations were seen in a similar frequency as describe in previous LA countries. For post antiEGFR TKI patients, plasma samples were the preferred method to evaluate T790M mutations with an adequate sensibility.

Using a multiplexed immunofluorescence approach to compare immune cell populations in subtypes of non-small cell lung cancer.

e20702 Background: Non-small cell lung cancer (NSCLC) accounts for approximately 80-85% of all lung cancer cases, and is characterized by a poor response to chemotherapy and a low survival rate. While immunotherapy has recently emerged as a successful approach in the treatment of NSCLC patients only 20-25% of patients show a positive response to anti-PD/PDL1 therapy, stressing the urgent need for additional immunotherapy options. NSCLC is a very heterogeneous disease with the two most common types being adenocarcinoma (ADCA) and squamous cell carcinoma (SCC). While a study using flow cytometry to analyze NSCLC on protein level reported the immune cell composition to be fundamentally different in ADCA compared with SCC (Daraselia et al. Am J Cancer Res, 2012 2(1):93), to the best of our knowledge this study will be the first of its kind performing an extensive analysis of NSCLS immune cell composition in tissue. Methods: In order to perform this comprehensive immunoprofiling of the NSCLC subtypes ADCA and SCC we will be using MultiOmyx™, an immunofluorescence (IF) multiplexing assay with similar staining characteristics as standard IHC stains but with the significant advantage that up to 60 protein biomarkers can be interrogated from a single FFPE section. Proprietary cell segmentation algorithms generate unique IDs for every cell and deep learning based cell classification algorithms then identify positive cells for each biomarker. Results: Using a 16-marker panel consisting of CD3, CD4, CD8, FoxP3, CD20, CD68, CD163, CD15, PD1, PDL1, ICOS, OX40, LAG-3, TIM-3, and PanCK we will analyze the proportion of B cells, T cell subtypes, M1/M2-type tumor-associated macrophages, as well as the expression of PD1/PDL1, in addition to novel immunotherapy targets LAG3, TIM3, ICOS, and OX40 in 20 samples from patients with NSCLC (10 ADCA and 10 SCC). Finally, from the same FFPE blocks that we will use to profile the immune cell composition on protein level we will also be extracting DNA/RNA in order to perform T cell receptor (TCR) sequencing, which will provide additional information on the diversity of T cells present in these tumors. Conclusions: It is our hope that these data will help provide new insights into the biology of ADCA and SCC that can ultimately be used to explore novel immunotherapeutic interventions for lung cancer treatment.

An optimized workflow for a fast molecular diagnosis of non-small cell lung cancer.

e13139 Background: Lung cancer is the most common and lethal malignancy throughout the world, in which non-small cell lung cancer (NSCLC) is the dominant subtype. To distinguish between different cell types of NSCLC, i.e. adenocarcinoma or squamous cell carcinoma, the morphologic features of cancer cells are important, as well as specific molecular markers, i.e. TTF1, p63 and p40. Genomic studies have revealed different genetic aberrations between specific subtypes of NSCLC, and distinct response to targeted therapy. Therefore, molecular testing is essential for precision diagnosis and personalized treatment. The canonical workflow from diagnosis to next generation sequencing (NGS) tests needs about two weeks. Importantly, sometimes the residue material left after diagnostic staining may not be enough for NGS tests. This means a secondary biopsy is needed, which is not patient friendly. Methods: We optimized the canonical workflow by using BIOCO, a magnetic collector attached to microtome knife holder that can collect the abandoned tissue during slicing FFPE blocks. Therefore, the material used for NGS test was collected simultaneous during slicing for pathological diagnosis. In total 123 FFPE tumor blocks from suspicious NSCLC patients were included to evaluate: 1. how long it will take from diagnosis to make treatment decision? 2. If the material collected by BIOCO is sufficient for NGS test and whether the result can be trusted. Results: 1. The BIOCO workflow only needs 7 days for the diagnosis and making therapeutic plan. 2. The BIOCO could collect enough FFPE tissue for NGS test even with HE, IHC stanning and FISH test for all the samples; however, 14 samples failed NGS test because of insufficient material left following the canonical workflow. 3. the detection sensitivity and specificity of the BIOCO method are comparable with the canonical workflow, with a consistence of 94.9%. Conclusions: Here we propose an optimized workflow by using the BIOCO collector we invented, which shortens the canonical workflow from two weeks to one week, and it fully utilizes the limited biopsy tissue for NGS testing so that the secondary biopsies are avoided.

Multi-subtype classification model for non-small cell lung cancer based on radiomics: SLS model.

Histological subtypes of non-small cell lung cancer (NSCLC) are crucial for systematic treatment decisions. However, the current studies which used noninvasive radiomic methods to classify NSCLC histology subtypes mainly focused on two main subtypes: squamous cell carcinoma (SCC) and adenocarcinoma (ADC), while multi-subtype classifications that included the other two subtypes of NSCLC: large cell carcinoma (LCC) and not otherwise specified (NOS), were very few in the previous studies. The aim of this work was to establish a multi-subtype classification model for the four main subtypes of NSCLC and improve the classification performance and generalization ability compared with previous studies. In this work, we extracted 1029 features from regions of interest in computed tomography (CT) images of 349 patients from two different datasets using radiomic methods. Based on "three-in-one" concept, we proposed a model called SLS wrapping three algorithms, synthetic minority oversampling technique, ℓ2,1-norm minimization, and support vector machines, into one hybrid technique to classify the four main subtypes of NSCLC: SCC, ADC, LCC, and NOS, which could cover the whole range of NSCLC. We analyzed the 247 features obtained by dimension reduction, and found that the extracted features from three methods: first order statistics, gray level co-occurrence matrix, and gray level size zone matrix, were more conducive to the classification of NSCLC subtypes. The proposed SLS model achieved an average classification accuracy of 0.89 on the training set (95% confidence interval [CI]: 0.846 to 0.912) and a classification accuracy of 0.86 on the test set (95% CI: 0.779 to 0.941). The experiment results showed that the subtypes of NSCLC could be well classified by radiomic method. Our SLS model can accurately classify and diagnose the four subtypes of NSCLC based on CT images, and thus it has the potential to be used in the clinical practice to provide valuable information for lung cancer treatment and further promote the personalized medicine.

FAM83A and FAM83B as Prognostic Biomarkers and Potential New Therapeutic Targets in NSCLC.

Although targeted therapy has improved the survival rates in the last decade, non-small-cell lung cancer (NSCLC) is still the most common cause of cancer-related death. The challenge of identifying new targets for further effective therapies still remains. The FAMily with sequence similarity 83 (FAM83) members have recently been described as novel oncogenes in numerous human cancer specimens and shown to be involved in epidermal growth factor receptor (EGFR) signaling. Here, gene expression of FAM83A and B was analyzed in a cohort of 362 NSCLC patients using qPCR. We further investigated relations in expression and their prognostic value. Functional assays in NSCLC cell lines were performed to evaluate FAM83A and B involvement in proliferation, anchorage-independent growth, migration, and the EGFR pathway. We observed a highly increased gene expression level of FAM83A (ø = 68-fold) and FAM83B (ø = 20-fold) which resulted in poor survival prognosis (p < 0.0001 and p = 0.002). Their expression was influenced by EGFR levels, pathway signaling, and mutation status. Both genes affected cell proliferation, and FAM83A depletion resulted in reduced migration and anchorage-independent growth. The results support the hypothesis that FAM83A and B have different functions in different histological subtypes of NSCLC and might be new therapeutic targets.

Clinical features and therapeutic options in non-small cell lung cancer patients with concomitant mutations of EGFR, ALK, ROS1, KRAS or BRAF.

BackgroundAlthough oncogenic driver mutations were thought to be mutually exclusive in non‐small cell lung cancer (NSCLC), certain tumors harbor co‐occurring mutations and represent a rare molecular subtype. The evaluation of the clinical features and therapeutic response associated with this NSCLC subtype will be vital for understanding the heterogeneity of treatment response and improving the management of these patients.MethodsThis retrospective study included 3774 samples from patients diagnosed with NSCLC. All samples were screened for EGFR, ALK, ROS1, KRAS, and BRAF mutation using the amplification‐refractory mutation system. The relationship between concomitant driver mutations and clinicopathologic characteristics, and patient clinical outcomes were evaluated.ResultsSixty‐three (1.7%) samples had more than one driver gene mutation. Among these, 43 were coalterations with an EGFR mutation, 20 with an ALK rearrangement, and eight with an ROS1 rearrangement. Except for ROS1 concomitant mutations that were more frequent in male patients (87.5%, P = 0.020), the clinicopathological features of the concomitant mutation patients were not significantly different from those harboring a single EGFR, ALK, or ROS1 mutation. Furthermore, first‐line EGFR‐TKI treatment did not significantly improve the progression‐free survival (PFS) of patients harboring EGFR concomitant mutation, compared to patients harboring a single EGFR mutation. However, for EGFR concomitant mutation patients, TKI therapy was more effective than chemotherapy (median PFS of 10.8 vs 5.2 months, P = 0.023). Lastly, KRAS mutations did not influence the EGFR‐TKI therapy treatment effect.ConclusionIn this study, concomitant mutations were found in 1.7% of the NSCLC. EGFR‐TKI therapy was more effective than chemotherapy for patients harboring EGFR concomitant mutation, and ROS1 concomitant mutations were more frequent in male patients. For patients harboring coalterations with an ALK or ROS1 rearrangement, we should be cautious when considering the therapeutic options.

Serum miR-210-3p as a Potential Noninvasive Biomarker of Lung Adenocarcinoma: A Preliminary Study.

Background: Development of noninvasive biomarkers could potentially contribute to extending the 5-year overall survival rate of nonsmall cell lung cancer (NSCLC) patients. Circulating microRNAs (miRNAs), due to their high stability, have the potential to become valuable cancer biomarkers. Methods: Using reverse transcription-quantitative polymerase chain reaction and testing three methods for data normalization, the expression levels of six miRNAs were evaluated in serum samples obtained from 50 NSCLC patients. Subsequently, the clinical significance of the tested miRNAs was determined. Results: Significant downregulation of miR-21-5p, miR-30a-5p, and miR-126-3p and upregulation of miR-210-3p and miR-486-5p in serum samples of NSCLC patients were identified in comparison to healthy controls. miR-205-5p appeared to be undetectable in all tested samples. Furthermore, miR-210-3p was differentially expressed between two subtypes of NSCLC. Receiver operating characteristic analysis for miR-210-3p revealed the area under the curve of 0.842 (95% confidence interval, 0.72-0.96; p = 0.0003) and demonstrated that miR-210-3p displayed considerable accuracy in discriminating between lung adenocarcinoma (AC) patients and healthy controls. Conclusions: Findings from this preliminary study indicate that five of six tested miRNAs were deregulated in the serum of NSCLC patients. Moreover, miR-210-3p appears to be a promising biomarker for diagnosis of lung AC.

Clinical associations and prognostic value of site-specific metastases in non-small cell lung cancer: A population-based study.

The prognosis of non-small cell lung cancer (NSCLC) is poor, particularly for patients with metastatic disease. Numerous efforts have been made to improve the prognosis of these patients; however, only a small number of studies have explored the occurrence rate and prognostic value of different patterns of distant metastasis (DM) in NSCLC systematically. To investigate these, information from patients diagnosed with NSCLC between 2010 and 2014 was collected from the Surveillance, Epidemiology and End Results database. Survival rate comparisons were performed using Kaplan-Meier analysis and log-rank tests. A Cox proportional hazard model was established to determine factors associated with improved overall survival (OS) and cancer-specific survival (CSS). The present study revealed that the most common site of single metastasis occurrence was bone, and the least common was the liver for NSCLC. As for multi-site metastases, the most common two-site metastasis involved bone and lung, and the most common three-site metastasis involved bone, liver and lung. As for NSCLC subtypes, large cell carcinoma (LCC) exhibited more specific metastatic features. The most common single metastatic site was the brain for patients with LCC, and the most common two-site metastatic combination was bone and liver. Patients with isolated liver metastasis exhibited the worst OS and CSS among patients with single metastasis. Furthermore, for patients with multi-site metastases, metastases involving the liver were associated with the worst OS and CSS among various combinations. To the best of our knowledge, the present study is the first to investigate the occurrence rate and prognostic value of different metastatic patterns of site-specific DM for NSCLC using a large population-based dataset. The findings of the present study may have vital implications for classifying patients with advanced NSCLC, thus laying a foundation for individualized precise treatment.

Metformin improves survival in lung cancer patients with type 2 diabetes mellitus: A meta-analysis

Background and objectiveIn recent years, many studies have investigated metformin and its effects on lung cancer. However, since previous studies have shown that the relationship between metformin and lung cancer is complicated, we performed a meta-analysis to analyze this relationship. Material and methodsAn electronic database search was conducted using PubMed, Embase, and Cochrane library. Outcomes were quantified with hazard ratios and 95% confidence intervals to compare lung cancer survival in patients treated with or without metformin. ResultsTen studies, involving 4397 participants, were included. In the pooled analysis, we found that metformin treatment significantly improved the survival of lung cancer patients (hazard ratio=0.75, 95% confidence interval: 0.70–0.80; P<0.001). Subgroup analysis showed that when stratified by geographic region, the hazard ratios for overall survival were 0.76 (95% confidence interval: 0.71–0.81, P<0.001) and 0.51 (95% confidence interval: 0.25–0.78, P<0.001) for Western and Asian countries, respectively. When stratified by lung cancer subtype, the hazard ratios for overall survival were 0.78 (95% confidence interval: 0.71–0.84, P<0.001), 0.73 (95% confidence interval: 0.66–0.81, P<0.001), and 0.51 (95% confidence interval: 0.25–0.78, P<0.001) for non-divided, non-small cell, and small-cell lung cancer subtypes, respectively. When stratified by study design, the hazard ratios for overall survival were 0.77 (95% confidence interval: 0.71–0.83, P<0.001) and 0.71 (95% confidence interval: 0.63–0.80, P<0.001) for cohort-based and case–controlled studies, respectively.